Modular Functional Peptides for the Intracellular Delivery of Nanoparticles

ABSTRACT

Described are peptides for delivery of a nanoparticle to the cytosol, the peptide comprising: (a) a nanoparticle association domain; (b) a proline-rich spacer domain; (c) an uptake domain; and (d) a vesicle escape domain comprising a non-hydrolyzable lipid moiety, wherein the spacer domain is between the nanoparticle association domain and the uptake and vesicle escape domains, and wherein the peptide, when attached to an extracellular nanoparticle, is effective to induce uptake of the nanoparticle by a cell and delivery of the nanoparticle to the cytosol of the cell. Also described are methods of delivery of a nanoparticle to the cytosol of a cell, the method comprising providing to a cell a nanoparticle attached to such a peptide. Exemplary nanoparticles include quantum dots.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/253,921 filed on Oct. 22, 2009, which is incorporatedherein by reference.

This application is also related to U.S. patent application Ser. No.12/606,766 filed on Oct. 27, 2009, which is incorporated herein byreference.

BACKGROUND

Quantum dots (QDs) provide many advantageous features that include highquantum yield, broad absorption spectra, large achievable Stokes shifts,narrow symmetric, size tunable emission spectra, and exceptionalresistance to photo- and chemical degradation, making them attractivereagents for the long-term visualization of cellular structures andprocesses. See references 1-4.

The methods employed to date for the intracellular delivery of QDs orother nanoparticles (NPs) can be grouped into three generalizedcategories based on their physicochemical nature. Passive delivery is anonspecific process that relies on the inherent physicochemicalproperties of the QD (surface charge and/or functionalization) tomediate uptake. Facilitated delivery utilizes a delivery agent (e.g., acationic peptide or a polymer) that is covalently attached to orelectrostatically complexed with the QDs to specifically induceinternalization. Both these techniques, while noninvasive, typicallyutilize the endocytic pathway which results in encapsulation of the QDswithin intracellular endolysosomal vesicles and thus requires furtherstrategies to liberate the sequestered QDs to the cytosol if that isultimately desired. Examples methods of facilitated delivery includeusing additional chemicals such as sucrose or chloroquine or addingpolymers such as polyethyleneimine during delivery to disrupt theendosomes by osmotic shock: such methods are generally quite toxic.Lastly, active delivery methods such as electroporation andmicroinjection deliver QDs directly to the cytosol through physicalmanipulation of the cell. However, these are highly invasive techniquesthat can often compromise the integrity of cellular structures andsubstantially reduce cellular viability (see reference 10). Thus, eachof the previously described methods for delivery of nanoparticles(including quantum dots) is deficient in some way.

Described herein are improvements in intracellular delivery ofnanoparticles.

BRIEF SUMMARY

In one embodiment, a peptide for delivery of a nanoparticle to thecytosol comprises: (a) a nanoparticle association domain; (b) a spacerdomain; (c) an uptake domain; and (d) a vesicle escape domain comprisinga non-hydrolyzable lipid moiety, wherein the spacer domain is betweenthe nanoparticle association domain and the uptake and vesicle escapedomains, and wherein the peptide, when attached to an extracellularnanoparticle, is effective to induce uptake of the nanoparticle by acell and delivery of the nanoparticle to the cytosol of the cell.

In a further embodiment, the spacer comprises from between 6 and 15proline residues, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15proline residues.

In another embodiment, a nanoparticle is delivered to the cytosol of acell by providing to a cell with a nanoparticle attached to such apeptide.

Exemplary nanoparticles include quantum dots.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows spectral properties and schematic of QD conjugates examinedherein. FIG. 1A shows normalized absorbance and emission of 510, 550 nmQDs and AlexaFluor 647 (AF647). FIG. 1B shows schematics of various QDconjugates.

FIG. 2 shows QD-CPP internalization and colocalization over time. Cellswere washed, fixed and stained with DAPI in FIG. 2A or supplied withfresh media and cultured for 4, 24 or 72 h prior to fixation andDAPI-staining in FIG. 2B. In FIG. 2A the DAPI, QD and marker signals areshown individually and merged while in FIG. 2B only the merged imagesare shown. Arrows indicate areas of colocalization. The scale bar is 10μm.

FIG. 3 shows the intracellular stability of polyhistidine-QDassociation. For FIG. 3A 510 nm dihydrolipoic acid (DHLA) QDs wereappended with 25 CPP to mediate uptake in HEK 293T/17 cells and ˜2Cy3-labeled peptides to monitor the FRET between the QD and the Cy3 dyeover time. FIG. 3B shows merged images of DAPI-stained nuclei, QD andCy3 signals at 1, 4, 24 and 72 h after conjugate delivery, where thescale bar is 10 μm. FIG. 3C shows the calculated Cy3/QD emission ratiois shown plotted as a function of time after initial conjugate delivery.FIG. 3D shows observed fluorescence for transferrin-Cy3 conjugatesdelivered alone (no QDs) is plotted as a function of time. In FIGS. 3Cand 3D the data points were fitted with a dose response logistic curvefit function.

FIG. 4 shows cellular delivery of QDs using various active andfacilitated methods. 510 nm DHLA-PEG QDs at a concentration of 400-500nM were delivered to HEK 293T/17 cells by electroporation in FIG. 4A ornucleofection in FIG. 4B. In FIG. 4C 510 nm DHLA-PEG QDs (800 nM) weredelivered using the pinocytic reagent, Influx™. 520 nm QDs (75 nM)capped with a 1:1 mixed surface of DHLA:DHLA-PEG were delivered to HEK293T/17 cells using Lipofectamine-2000™ in FIG. 4D or the branchedpolymer, polyethyleneimine in FIG. 4E. The images in FIGS. 4D and 4Eshow merged images of the QD signal and the fluorescence from theendocytic marker, AlexaFuor647-transferrin. In FIGS. 4A through E thecells were imaged after fixation and staining with DAPI. In FIG. 4F 550nm DHLA-PEG QDs (100 nM) appended with 25 CPP were coincubated with 100μM pyrenebutyrate in PBS for 30 min at 37° C. prior to washing andimaging of live cells (no DAPI present). The scale bar is 10 μm.

FIG. 5 shows cellular delivery and cytotoxicity of PULSin™-QDconjugates. 520 nm 1:1 DHLA:DHLA-PEG mixed surface QDs (100 nM final QDconcentration) were complexed with PULSin™ polymer and incubated for 1-3h with COS-1 cells A or HEK 293T/17 cells B. Shown are images in whichthe DAPI, QD and AlexaFluor 647-transferrin images are merged at 1d, 2d,3d, 4d and 5d post-QD delivery. Regular arrows denote areas ofcolocalization between the QDs and endosomes (a yellow color was noted,indicating merged QD and transferrin signals). Open circle-terminatedarrows indicate areas where the QD signal is separated from theendosomal marker. The scale bar is 10 μm. Cytotoxicity datademonstrating the effects of the PULSin™-QD complexes on cellularproliferation are shown for COS-1C and HEK 293T/17 cells D. Cells wereincubated with the complexes for 3 h, washed and subsequently culturedfor 72 h prior to viability assay. When the QDs are present, theconcentration given is that of the QDs. Each data point represents themean±SD of triplicate measurements

FIG. 6 shows the cellular delivery and cytotoxicity of Palm-1 peptide(SEQ ID No: 3) conjugates with quantum dots. 550 nm DHLA-PEG QDs weredecorated with the Palm-1 peptide, incubated for 1-2 h along withAlexaFluor 647-transferrin, and imaged at 48 h post-delivery withinCOS-1 in FIG. 6A or HEK 293T/17 cells in FIG. 6B. The inset in themerged images shows a single cell with the cell membrane highlighted forclarity. FIGS. 6C and 6D show corresponding cytotoxicity data for theinhibition of cellular proliferation by QD-Palm-1 complexes in COS-1 andHEK 293T/17 cells, respectively. Cells were incubated with the materialsfor 1 h, washed, and subsequently cultured for 72 h prior to viabilityassay. When the QDs are present, the concentration given is that of theQDs. Each data point represents the mean±SD of triplicate measurements.

FIG. 7 illustrates the functionalization of NPs with modularmultifunctional peptides. FIG. 7A shows a general schematic of anexemplary NP-peptide assembly. The schematic peptide shown consists ofmultiple functional domains: a NP association domain (1); a spacerdomain (2); and multiple domains that impart biological activity (3 and4). FIG. 7B shows a QD-peptide assembly for intracellular uptake andendosomal escape. The modular peptide consists of a hexa-histidine (H6)domain for assembly to the QD surface, a proline-rich spacer domain(P9GG), a positively charged lysine-rich domain (VKIKK, SEQ ID No: 7)for cellular association and uptake, and a synthetic palmitic acid group((Pal)Dap) for cell membrane association and insertion and vesicleescape.

FIG. 8 illustrates a model of NP-peptide assemblies.

FIG. 9 illustrates the cellular uptake and intracellular fate of variousQD-peptide assemblies. In FIG. 9A the assembles have Palm-1b, in FIG. 9BPalm-2b, and in FIG. 9C Palm-3b.

FIG. 10 illustrates various moieties employed in the peptides describedherein. “Dab” represents diaminobutyric acid.

DETAILED DESCRIPTION

This invention is not limited to the particular methodology, devices,solutions or apparatuses described, as such methods, devices, solutionsor apparatuses can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention. For instance, although several examples use quantum dots, oneof ordinary in the art would understand that similar techniques can beused with other nanoparticles as described below.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a nanoparticle” includes a plurality of suchnanoparticles.

Terms such as “connected,” “attached,” “linked,” and “conjugated” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each sub-range between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed. Where a value being discussed has inherentlimits, for example where a component can be present at a concentrationof from 0 to 100%, or where the pH of an aqueous solution can range from1 to 14, those inherent limits are specifically disclosed. Where a valueis explicitly recited, it is to be understood that values which areabout the same quantity or amount as the recited value are also withinthe scope of the invention. Where a combination is disclosed, eachsubcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention. Where any element ofan invention is disclosed as having a plurality of alternatives,examples of that invention in which each alternative is excluded singlyor in any combination with the other alternatives are also herebydisclosed; more than one element of an invention can have suchexclusions, and all combinations of elements having such exclusions arehereby disclosed.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of amino acids linked through peptide bonds. The termsdo not refer to a specific length of the product. Thus, “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The terms include polypeptides containingpost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and sulphations. Inaddition, protein fragments, analogs (including amino acids not encodedby the genetic code, e.g. homocysteine, ornithine, D-amino acids, andcreatine), natural or artificial mutants or variants or combinationsthereof, fusion proteins, derivatized residues (e.g. alkylation of aminegroups, acetylations or esterifications of carboxyl groups) and the likeare included within the meaning of polypeptide. The peptides that can beused as described herein include multiple amino acids, which may benatural, synthetic or a mixture thereof. Each peptide may expressdifferent side chains, if desired. Other amide oligomers such as betapeptides, peptoids and peptide nucleic acids may also be used.

The term “proline-rich spacer” as used herein refers to a spacercomprising at least about 50% proline residues, optional at least 60%,70%, 80%, or 90% proline residues, or optionally entirely prolineresidues. Preferably, the spacer has a length of from six to eighteenresidues.

The term “non-hydrolyzable” as used herein refers to a moiety thatremains substantially not hydrolyzed by the normal action ofintracellular enzymes, so that it remains attached to a peptide. Asdescribed in more detail below, it is believed that the property ofbeing non-hydrolyzable contributes to escape from the endosomal systeminto the cytosol.

The terms “semiconductor nanocrystal,” “SCNC,” “SCNC nanocrystal,”“quantum dot,” and “QD” are used interchangeably herein and refer to aninorganic crystallite of about 1 nm or more and about 1000 nm or less indiameter or any integer or fraction of an integer therebetween,preferably at least about 2 nm and about 50 nm or less in diameter orany integer or fraction of an integer therebetween, more preferably atleast about 2 nm and about 20 nm or less in diameter (for example about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nm). QDs are characterized by their uniform nanometer size.

A QD is capable of emitting electromagnetic radiation upon excitation(i.e., the QD is luminescent) and includes a “core” of one or more firstsemiconductor materials, and may be surrounded by a “shell” of a secondsemiconductor material. A QD core surrounded by a semiconductor shell isreferred to as a “core/shell” QD. The surrounding “shell” material willpreferably have a bandgap energy that is larger than the bandgap energyof the core material and may be chosen to have an atomic spacing closeto that of the “core” substrate.

The core and/or the shell can be a semiconductor material including, butnot limited to, those of the groups II-VI (ZnS, ZnSe, ZnTe, US, CdSe,CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe,SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb,InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like)materials, PbSe, and an alloy or a mixture thereof. Preferred shellmaterials include ZnS.

A QD is optionally surrounded by a “coat” of an organic capping agent.The organic capping agent may be any number of materials, but has anaffinity for the QD surface. In general, the capping agent can be anisolated organic molecule, a polymer (or a monomer for a polymerizationreaction), an inorganic complex, or an extended crystalline structure.The coat can be used to convey solubility, e.g., the ability to dispersea coated QD homogeneously into a chosen solvent, functionality, bindingproperties, or the like. In addition, the coat can be used to tailor theoptical properties of the QD.

Thus, the quantum dots herein include a coated core, as well as acore/shell QD.

The term “nanoparticle” as used herein includes the above-mentioned QDsin addition to other nano-scale and smaller particles such as carbonnanotubes, proteins, polymers, dendrimers, viruses, and drugs. Ananoparticle has a size of less than about 1 micron, optionally lessthan about 900, 800, 700, 600, 500, 400, 300, 100, 80, 60, 50, 40, 30,20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nanometers. A nanoparticle may havevarious shapes such as a rod, a tube, a sphere, and the like.Nanoparticles may be made from various materials including metals,carbon (such as carbon nanotubes), polymers, and combinations thereof. Ananoparticle for cytosolic delivery by a peptide may be referred to as acargo or payload.

The QDs as well as other nanoparticles, may be biofunctionalized for usein, for example, in vivo tissue and cellular labeling, development ofbiological labels based on quantum dot probes and biosensor development.Other uses of the biofunctionalized materials may include proteinordering for molecular electronics where quantum dots could serve asfluorophores and electronic components, energy harvesting, quantum dotbased bar coding, and drug discovery assays where the fluorescenceproperties of the quantum dots may be combined with bioactive peptides.

The functionalization may be used to impart a variety of properties toquantum dots and/or nanoparticles including, but not limited to, theability to homogeneously disperse the quantum dots and/or nanoparticlesin buffer solutions and a variety of polar solvents at various pHvalues; biocompatibility; biotargeting by allowing the use ofpeptide-driven binding to specific cell receptors such as the TATsequence; providing specific points of modification directly on thequantum dot substrate by using, for example, amine groups for reactingwith N-hydroxysuccinimide esters; providing bio-recognized sequencessuch as the AviTag sequence which is specifically biotinylated as anexample; providing protease-recognized cleavage sites; providingpolyhistidines for metal affinity coordination; and providing functionalgroups for further targeted modification, including, for example, aminogroups, carboxyl groups, azide groups, alkyne groups, hydrazine groups,aldehyde groups, aminooxy groups, ketone groups, maleimide groups orthiol groups for dye/quencher or other chemical modification steps.

Materials. Sucrose, chloroquine, and polyethyleneimine (PEI, 25 kDa Mw,branched polymer) were purchased from Sigma (St. Louis, Mo.). Influx™reagent, Lipofectamine-2000™ and the subcellular organelle markersAlexaFluor 647-transferrin (λabs 650 nm/λem 668 nm), BODIPYTR-ceramide-BSA (λabs 589 nm/λem 616 nm), LysoTracker Red DND-99 (λabs577 nm/λem 590 nm) and the nuclear stain DAPI (λabs 350 nm/λem 450 nm)were obtained from Invitrogen (Carlsbad, Calif.). PULSin™ was purchasedfrom Polyplus-transfection (New York, N.Y.). Other materials wereobtained as described.

Quantum Dots. CdSe—ZnS core-shell QDs with emission maxima centered at510, 520, or 550 nm were synthesized and made hydrophilic by exchangingthe native trioctylphosphine/trioctylphosphine oxide (TOP/TOPO) cappingshell with either DHLA (dihydrolipoic acid) or polyethylene glycol (PEG)appended DHLA ligands as described previously (see references 17 and18). These are subsequently referred to herein as DHLA or DHLA-PEGligands. In general, PEGylated-QDs are preferred as they providesuperior intracellular solubility and pH stability; however, some of thedelivery reagents utilized required QDs with charged surfaces to mediateelectrostatic interactions. 510 nm QDs capped with either DHLA orDHLA-PEG ligands were used for CPP-mediated delivery. The 520 nm QDsdelivered by PULSin™, PEI or Lipofectamine-2000™ were capped with a 1:1ratiometric mix of DHLA and DHLA-PEG ligands. 550 nm QDs used forelectroporation, nucleofection, Influx™- and peptide-mediated deliverywere capped with DHLA-PEG.

FIG. 1A shows normalized absorbance and emission of 510, 550 nm QDs andAlexaFluor 647 (AF647). FIG. 1B is a schematic of QD conjugates forfacilitated QD delivery. CdSe—ZnS core-shell QDs capped with eithercharged DHLA or neutral DHLA-PEG ligands are noncovalently associatedwith linear cationic polymers, cationic liposomes or histidine-taggedpeptides to mediate QD endocytosis. The sequences of the CPP, NLS, andPalm-1 peptides used herein are also shown. In FIG. 1, “Aib” refers toalpha-amino isobutyric acid and “Pal” refers to a nonhydrolyzablepalmitate group that is covalently attached to a diaminopropionic acidfunctionality synthesized into the peptide backbone.

Peptides. The cell-penetrating peptide (CPP) having the sequenceR9GGLA(Aib)SGWKH6 (SEQ ID No: 1) was used. “Aib” represents theartificial residue alpha-amino isobutyric acid. The polyarginine tract(R9) mediates cellular uptake and is separated from a polyhistidinetract (H6) for assembly to the QD surface by a linker domain(GGLA(Aib)SGWK). The nuclear localization signal (NLS) containingpeptide was synthesized with the sequence H6WGLA(Aib)SGPKKKRKV(SEQ IDNo: 2). The palmitoylated peptide (Palm-1) sequence wasWG(Pal)VKIKKP9GGH6 (SEQ ID No: 3) where “Pal” corresponds todiaminopropionic acid, which is a nonhydrolyzable palmitate group thatis covalently attached to a diaminopropionic acid functionality, and inthis example synthesized into the peptide backbone. The initialtryptophan residue in Palm-1 provides an aromatic ring structure formonitoring by absorption spectroscopy, and it not believed to be crucialto the function of the peptide. A nonspecific peptide with the sequenceH6SLGAAAGSGC (providing a general structure of H6-spacer-cysteine) (SEQID No: 8) was labeled with Cy3-maleimide (λabs 550 nm/λem 570 nm, GEHealthcare, Piscataway N.J.) on the terminal cysteine residue and usedfor FRET studies. The peptides were synthesized using Boc-solid phasepeptide synthesis, purified by HPLC, and characterized by electrosprayionization mass spectrometry (see references 13 and 19). Preparation ofthe nonhydrolyzable palmitate group that is covalently attached to adiaminopropionic acid functionality synthesized into the peptidebackbone can be accomplished by one of ordinary skill in the art, asdescribed in reference 48.

Cell Culture. Human embryonic kidney (HEK 293T/17) and African greenmonkey kidney (COS-1) cell lines (ATCC, Manassas, Va.) were cultured incomplete growth medium (Dulbecco's Modified Eagle's Medium (DMEM;purchased from ATCC)) supplemented with 1% (v/v) antibiotic/antimycoticand 10% (v/v) heat inactivated fetal bovine serum (ATCC). Cells werecultured in T-25 flasks and incubated at 37° C. under 5% CO2 atmosphereand a subculture was performed every 3-4 days (see reference 13).

Cellular Delivery of Quantum Dots. The QD delivery experiments wereperformed on adherent cells seeded into the wells of Lab-Tek 8-wellchambered #1 borosilicate coverglass (Nalge Nunc, Rochester, N.Y.)coated with 2 μg/mL fibronectin. For electroporation and nucleofection,adherent cells were harvested by trypsinization prior to performing QDdelivery to cells in suspension. Cells were then seeded to chamberedcoverglass wells and imaged after cell attachment. The endosomal markerAlexaFluor647-transferrin was included as indicated in the text. Forimaging, cells were washed with phosphate buffered saline (PBS, 137 mMNaCl, 10 mM phosphate, 3 mM KCl, pH 7.4), fixed with 3.7%paraformaldehyde in PBS and nuclei were stained with DAPI (Sigma) unlessotherwise indicated.

Comparative Example: Electroporation and nucleofection of QDs. Forelectroporation, cells were harvested by trypsinization and recoveredfor 1 hour in complete growth medium, pelleted, and resuspended in PBS.510 nm DHLA-PEG QDs (0.5 μM final concentration) were mixed with 1×104cells to a final volume of 100 μL in PBS in a 0.2 mm electroporationcuvette. The cuvette was subjected to 100 V for a 20 ms pulse using aGenePulser XCell electroporator (Bio-Rad, Hercules, Calif.). Cells wereresuspended in complete growth medium, seeded into fibronectin-coated8-well chambered coverglass, fixed and imaged after 24 hrs. Fornucleofection delivery, 1×10⁶ cells were harvested and resuspended in100 μL of NUCLEOFECTOR reagent (Amaxa, Gaithersburg, Md.). 510 nmDHLA-PEG QDs were included at a final concentration of 0.4 μM. Thecell/QD/reagent mixture was pulsed in the NUCLEOFECTOR using a presetprotocol adapted for each specific cell line (20, 21). Cells werecultured and imaged as described for electroporation. As a positivecontrol, a plasmid encoding monomeric red fluorescent protein (RFP) wasdelivered using the same conditions as those for QD delivery andpositive expression of the RFP confirmed.

Comparative Example: INFLUX-mediated delivery. The INFLUX cell-loadingreagent mediates the intracellular release of materials internalized viapinocytic vesicles by delivering the materials to cells in a hypertonicmedium followed by the transfer of the cells to a hypotonic medium toinduce vesicle disruption (see reference 22). 510 nm DHLA-PEG QDs werediluted to a final concentration of 300-800 nM in the suppliedhypertonic delivery media according to manufacturer's instructions andincubated on the cells for 30 min at 37° C. The hypertonic medium wasexchanged for hypotonic medium (serum-free culture medium diluted by 40%with deionized water) and incubated on the cells for 3 min. The cellswere then recovered in complete growth medium for 30 min and culturedfor various lengths of time prior to washing and fixation for imaging.

Comparative Example: Lipofectamine 2000™-mediated delivery. 520 nm QDswith a 1:1 mixed surface of DHLA:DHLA-PEG were incubated withLIPOFECTAMINE 2000 for 20 min in serum free medium at a ratio of 1 μLLIPOFECTAMINE 2000 per 1.5 μmol QD. The complexes, at a final QDconcentration of 75 nM, were incubated with the cells for 4 hours,removed and replaced with complete growth medium. The cells were thencultured for 24-36 h prior to washing and fixation.

Comparative Example: Polyethyleneimine-mediated delivery. PEI wasincubated with 520 nm mixed surface DHLA:DHLA-PEG QDs in serum freemedium at a ratio of 10 μg PEI per 1 pmol QD for 15 min. This ratio waspreviously determined in delivery experiments to yield maximal QDuptake. The complexes were then diluted into serum free media to a finalQD concentration of 75-100 nM, incubated with cells for 2 h and removed.Cells were then cultured in complete media for 24-36 h, washed andfixed.

Comparative Example: PULSin™-mediated delivery. A stock solution of 520nm DHLA:DHLA-PEG QDs (1 μM in 0.1 M borate buffer, pH 8.9) was dilutedto 0.5 μM in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,pH 8.2). PULSin™ delivery reagent was added (1 μL per 20 μmol QD) andcomplex formation occurred for 20 min at 25° C. The complexes werediluted into serum free medium to a final QD concentration of 100 nM andincubated on cells for 1-3 h after which complexes were removed andreplaced with complete growth medium. Cells were subsequently culturedfor up to 5 days to monitor intracellular QD distribution.

Inventive Example: Peptide-mediated delivery. QD-CPP, QD-NLS andQD-Palm-1 bioconjugates were formed by diluting a stock solution ofpreformed peptide-QD complexes (1 μM QD assembled with 25 CPP, 30 NLS or75 Palm-1 peptides per QD in 0.1 M borate buffer, pH 8.9) into completegrowth medium to a final QD concentration of 50-100 nM. These peptide:QDratios were determined experimentally for each peptide to be that ratiothat yielded a high degree of cell uptake. The self-assembledbioconjugates were then incubated with cells as described elsewhereherein. For monitoring the intracellular fate of QD-CPP assemblies,AlexaFluor 647-transferrin, LysoTracker Red DND-99 and BODIPYTR-ceramide-BSA subcellular markers were included at the manufacturer'srecommended concentrations. In some experiments, QD-CPP complexes werealso incubated with cells in the presence of pyrenebutyrate (100 μM),sucrose (500 mM) or chloroquine (500 μM) to test either membranetranslocation or to induce endosomal disruption and release of theinternalized QDs to the cytosol. The general scheme for the assembly ofQDs with cationic polymers, cationic amphiphiles or histidine-taggedpeptides is shown in FIG. 1B.

Microscopy and Image Analysis. The intracellular distribution of QDs wasanalyzed by differential interference contrast (DIC) and epifluorescencemicroscopy using an Olympus IX-71 total internal reflection fluorescencemicroscope equipped with a 60× oil immersion lens. Samples were excitedusing a Xe lamp and images were collected using standard filter sets forDAPI, FITC (for QDs), TRITC (for Cy3 and Texas Red) and Cy5 (forAF647-transferrin). Merged images were generated using Adobe PhotoShop.Förster resonance energy transfer (FRET) measurements for determiningthe intracellular stability of QD-peptide association over time wereperformed by imaging 510 nm donor QD-CPP conjugates decorated withapproximately 2 Cy3-labeled acceptor peptides per QD. The Förster radius(R0) for this donor-acceptor pair is approximately 47 Å. Side-by-sidesplit fluorescence images were collected and quantitated using aDualView system (Optical Insights, Tucson, Ariz.) equipped with a 565 nmdichroic filter. The QDs and Cy3 were excited at 488 nm and theirrespective emissions were separated with the dichroic filter anddeconvoluted. Signal intensities were measured at various time pointsover a three day period to calculate the Cy3/QD emission ratio (definedas [Cy3em/Cy3em+QDem]). This ratio corrects for any direct excitation ofthe Cy3 dye which may occur intracellularly (see reference 23). Tocorrect for any leakage of the QD signal into the Cy3 channel, thisratio was also calculated for cells exposed to QD-CPP alone (noCy3-labeled peptide) and subtracted from the Cy3/QD emission ratio togive the corrected ratio ([Cy3em/Cy3em+QDem]−QDem). A decrease in thisratio over time indicates either the dissociation of the Cy3-labeledpeptide from the QD surface or degradation of the Cy3 fluorophore.

Cytotoxicity Assays. Cellular toxicity was assessed using the CellTiter96 Cell Proliferation Assay (Promega, Madison Wis.) according to themanufacturer's instructions. This assay is based upon the conversion ofa tetrazolium substrate to a formazan product by viable cells at theassay end point (see reference 24). Cells (1×10⁴ cells/well) werecultured in 96-well microtiter plates in complete growth medium in thepresence of increasing concentrations of QDs, free peptide or polymer,or QDs in complex with peptide or polymer. In each case, the materialswere incubated with the cells for the time required for efficient QDuptake. The materials were subsequently replaced with complete growthmedium and the cells were cultured for 72 hours.

Long-term Intracellular Fate of QD-CPP Conjugates. The intracellularfate of the delivered QD-CPP complexes was followed at various timepoints in cell culture. HEK 293T/17 cells were incubated withDHLA-capped 510 nm QDs complexed with the CPP while counter-labelingeither the endosomes (AlexaFluor 647-transferrin), lysosomes(LysoTracker Red DND-99), or the Golgi complex (BODIPY TR-ceramide-BSA).As shown in FIG. 2A, one hour post-delivery the QDs adopted a punctate,vesicular appearance with QD fluorescence in green overlapping theendosome and lysosome markers, while no overlapping QD signal wasobserved with the Golgi complex markers. This confirms that the QD-CPPcomplexes were located within the endolysomal system in agreement withprevious results observed by the present inventors. Similar data wascollected for cells exposed to QD-CPP complexes and the same markers at4, 24 and 72 hours after delivery, as seen in FIG. 2B. After 3 days, theQD-CPP complexes still colocalized with the endolysosomal markersalthough it is noted that at the later time points, the QD distributionappears to be primarily perinuclear probably reflecting that these arelater or more ‘mature’ endosomes. When the cells were cultured beyond 3days, similar results were obtained. These results demonstrate thatwhile the CPP mediates the efficient uptake of the QDs, it does notfacilitate the release of the QDs to the cytosol over time. Similar datawere obtained with DHLA-PEG capped QDs showing that the nature of thecapping ligand, i.e., charged vs. neutral, had no effect onintracellular QD fate over time.

Intracellular Stability of QD-CPP Assemblies. For the successfulimplementation of QD-peptide and QD-protein conjugates in intracellularapplications, it is desirable for them to exhibit long-term stabilityduring and after uptake by the cell. For example, labeling specificsubcellular organelles such as mitochondria or the nucleus withQD-peptide conjugates requires the stable association of the targetingpeptide with the QD surface throughout the uptake and targeting process.Of particular interest in the case of the self-assembled QD-CPPdescribed herein (generated by polyhistidine-zinc interactions) is theconjugate stability within the endolysosomal vesicles during the threeday culture period. Given that the normal pKa of histidine residues is˜6.5 and that the pH of the vesicles can drop to as low as ˜5.0 to 5.5during the formation of late endosomes and lysosomes (see reference 28),protonation of the imidazole side chains of the polyhistidine tractcould result in dissociation of the CPP from the QD surface.Alternatively, several proteases including cathepsins and severalaspartate proteases are endogenously expressed in the endolysosomalsystem and these may also proteolyze the peptides (see reference 29).Thus, confirmation of the long-term intracellular stability of QD-CPPassemblies was warranted.

To investigate this issue, QD-peptide conjugates were prepared thatengaged in FRET and their intracellular interactions were monitored overtime. 510 nm DHLA-capped QDs were first assembled with an average of ˜2Cy3-labeled His₆-peptides and then the CPP was added to form the fullconjugate (schematic in FIG. 3A). Due to the peptide's small size andproximity of the Cy3 acceptor to the nanocrystal surface, this valenceresults in a ca. 40% quenching of the QD PL by FRET. The resultingconjugates were delivered to HEK 293T/17 cells and the cells werecultured for three days. QD-donor and Cy3-acceptor FRET interactionsover time within the cells were measured by exciting the QD andcollecting side-by-side split fluorescence images using the DualViewsystem and deconvoluting the subsequent intensity data. The excellentspectral separation (˜60 nm) between the QDs and Cy3 emission maximafacilitated this collection. FIG. 3B shows representative images inwhich the QD and Cy3 signals are merged at various time points duringthe culture period. A distinct one-to-one overlap in the punctatesignals of both QD and dye was observed demonstrating colocalization ofthe QDs and Cy3-labeled peptides within endocytic vesicles throughoutthe culture period. Analyses of the signals showed the pair was activelyengaged in FRET (close proximity) and not just present within the sameendosomal compartments. When the Cy3/QD emission ratio (normalized andcorrected for direct Cy3-acceptor excitation) was calculated a gradualdecrease was observed over time, culminating in a 36% decrease overthree days (FIG. 3C). A decrease in this ratio would result from eitherCy3-peptide dissociation from the QD surface (loss of His₆ interactionsor proteolysis) or by chemical degradation of the dye. Controlexperiments revealed that the latter scenario was the case. When Cy3 wasdelivered to the endosomes as a Cy3-labeled transferrin conjugate (noQDs present), the fluorescence output of the dye decreased approximately34% over the same time period, in excellent agreement with the decreaseobserved in the above QD-CPP-Cy3-labeled peptide constructs (FIG. 3D).Control experiments performed with QD donors alone showed no change inthe relative QD PL. These results suggest that the His₆-bearing peptidesremain stably conjugated to the QD Zn-surface, even within the acidicenvironment of the endocytic vesicles over time. This strong affinity,even at lower pH, appears to be as a result of cooperative interactionsresulting from the multiple histidine residues present on each peptideinteracting with the QD surface (see reference 14). Previously, thestability of the QD-peptide conjugates within endosomes had only beenverified for one hour after uptake using two-photon excitation FRETmicroscopy (see reference 23). As best can be determined, presentedherein represent the first instance in which the His-zinc interactionhas been shown to be stable intracellularly over three days. Thisfinding has important implications for the use of this QD conjugateassembly strategy in long-term intracellular labeling and imagingapplications.

Cytosolic Delivery of QDs. Having confirmed the long-term sequestrationof QDs within endosomes following CPP-facilitated uptake, a means bywhich to deliver QDs to the cytosol was sought. Thus began an exhaustiveinvestigation of methodologies to either: (1) deliver the QDs directlyto the cytosol via direct physical manipulation of the cell (activedelivery), or (2) decorate the QDs with peptides or polymers that couldmediate both endocytic uptake and the subsequent release of the QDs fromwithin endocytic vesicles (facilitated delivery followed by endosomalescape). The individual approaches are summarized in Table 1 and theresults of the various methods tested are discussed in the followingsections.

TABLE 1 Summary of QD Delivery Strategies Examined Herein DeliveryMethod/Agent Mechanism of Uptake Quantum Dot Fate and Toxicity ActiveDelivery Electroporation Membrane pore formation Poor uptake, QDsaggregated, toxic^(a) Nucleofection Membrane pore formation Poor uptake,QDs aggregated, toxic^(a) Facilitated Delivery Pinocytosis Influx ™Pinocytosis Endosomal, QDs punctate, moderately toxic^(a)Polymer-mediated Lipofectamine2000 ™ Endocytosis Endosomal, QDspunctate, toxic^(b) Polyethyleneimine Endocytosis Endosomal, QDspunctate, toxic^(a) PULSin ™ Endocytosis Cytosolic, QDs dispersed,toxic^(b) Peptide-mediated CPP peptide Endocytosis Endosomal, QDspunctate, minimally toxic^(b,c) NLS peptide Endocytosis Endosomal, QDspunctate, minimally toxic^(b) Palmitoylated peptide (Palm-1) EndocytosisCytosolic, QDs dispersed, minimally toxic^(b) Augmented peptide-mediatedCPP peptide + sucrose Endocytosis Cytosolic, QDs punctate, toxic^(a) CPPpeptide + chloroquine Endocytosis Cytosolic, QDs punctate, toxic^(a) CPPpeptide + pyrenebutyrate Membrane translocation Mixed membranous andendosomal, toxic^(a) ^(a)General toxicity assessment made by visualinspection of cellular morphology and rate of proliferation compared tocontrol cells during delivery experiments. ^(b)Toxicity measured by cellproliferation assay as described elsewhere herein. ^(c)Toxicity reportedin reference 13.

Active delivery. Electroporation and nucleofection are primarily usedfor the cellular delivery of nucleic acids and employ an externallyapplied electric field to increase the excitability and permeability ofthe membrane's phospholipid bilayer allowing charged extracellularmaterials to directly enter the cytosol during an electric pulse (seereference 30). Nucleofection further incorporates a proprietarytransfection reagent to mediate the subsequent localization ofinternalized materials to the nucleus (see references 20 and 21). Asshown in FIGS. 4A and 4B, when 510 nm DHLA-PEG capped QDs were deliveredto HEK 293T/17 cells using electroporation or nucleofection, theyadopted a punctate morphology indicative of QD aggregation within thecytosol. This delivery bypassed the endosomes as confirmed bytransferrin counterstaining. In contrast, when QDs capped with thesesame ligands were microinjected into cells, they adopted a highlydisperse staining across the entire cytosol for long periods of time(see reference 18). This indicates that the electric field and/orprocess itself adversely affects subsequent intracellular QD solubility.Further, no evidence of nuclear accumulation or localization within theperinuclear spaces was observed even after extended culture followingdelivery for both methods. Also noted was a high degree of cell deathand estimate that only 50% of cells remained viable following deliveryattempts.

Electroporation-based QD delivery has yielded similar results inprevious reports (see references 31 and 32). Cumulatively, the QDaggregation and subsequent high cellular morbidity suggest thatelectroporation and nucleofection are not effective means of deliverydespite their ability to deliver QDs directly to the cytosol.

Facilitated delivery. Facilitated delivery of QDs involves the use ofexogenous agents that are added to the extracellular medium or complexedwith the QDs to exploit the cell's innate processes of pinocytosis orendocytosis. Pinocytosis, or fluid-phase uptake, is a nonspecific formof endocytosis in which minute amounts of extracellular fluids andmaterials are internalized within small vesicles. Endocytosis, incontrast, is a specific process as the uptake of extracellular materialsis mediated by their interaction with cognate cell surface receptorsthat become spatially concentrated within the forming endocytictransport vesicles (see references 33 and 34). The exogenous agents forfacilitated delivery can take the form of chemicals or drugs that areco-incubated with the QDs during the delivery process or alternativelythey can be polymers or peptides that are either covalently attached ornon-covalently associated (electrostatically) with the QD surface.

Pinocytosis-mediated delivery. To exploit this process, the commercialpinocytosis reagent known as Influx™ was used. This reagent isco-incubated with the QDs and cells in a hypertonic medium whichpromotes the uptake of extracellular materials within pinocyticvesicles. The cells are then briefly incubated in a hypotonic medium toswell and disrupt the vesicles, releasing the internalized materialsinto the cytosol; in essence this is a modified intracellular osmoticshock protocol. A solution of 510 nm DHLA-PEG QDs was prepared in thehypertonic delivery media (including the Influx™ reagent) and incubatedwith HEK 293T/17 cells for the recommended period of time for uptake(˜30 min). As shown in FIG. 4C, only a modest degree of uptake occurredeven when the QD concentration was substantially increased to 800 nM.Further, the intracellular QD morphology remained punctate even afterseveral days, indicating their persistent localization within pinocyticvesicles. Jaiswal et al. also reported the long-term intracellularsequestration of pinocytically delivered QDs (see reference 6). In thatstudy, 400 to 600 nM negatively charged DHLA-capped CdSe/ZnS QDs wereincubated with HeLa cells for 2-3 hrs without any exogenous pinocytosisreagent present: using a plasmid-expressed fluorescent protein endosomalmarker for counter-labeling, the QDs remained trapped within vesiclesfor up to nine days in culture.

Polymer-mediated delivery. A number of commercial cationic polymers havebeen developed for gene delivery and transfection applications. Thesereagents self-assemble electrostatically to negatively-charged species(e.g., nucleic acids) while simultaneously mediating interactions of theresulting complex with the plasma membrane to induce endocytosis. Oncecompartmentalized within intracellular vesicles, it is believed that thecationic polymers can facilitate endosomal disruption via osmotic lysis(the ‘proton sponge’ effect), releasing the vesicle contents to thecytosol (see references 35 and 36). It was hypothesized that QDs bearinga net negative surface charge could complex with such polymers and thusutilized 520 nm QDs capped with a 1:1 mixed surface of DHLA:DHLA-PEGligands. This cap exchange strategy provides both a charged moiety(DHLA) along with the extended pH stability provided by the PEG ligands(see references 18 and 37). Initial experiments utilized the well-knowncationic liposomal transfection reagent Lipofectamine-2000™.QD-Lipofectamine complexes were formed as described in the Methodssection and incubated with HEK 293T/17 cells for 4 hours. As shown inFIG. 4D, it was found that the QDs were completely colocalized with theco-delivered transferrin endosomal marker. Extended monitoring of thecells for several days after delivery did not reveal any changes in thismorphology. Interestingly, Derfus et al., demonstrated that commercialPEG-coated QDs delivered to HeLa cells using this reagent appeared to belargely present within the cytosol (see reference 31). However, theirinterpretation was complicated by the fact that the QDs also formedaggregates of several hundred nanometers in diameter within the cytosoland were not well dispersed. It was not apparent if the liposomalpolymer was responsible for inducing the intracellular QD aggregation inthis case.

Polyethyleneimine (PEI) is a cationic polymer that is sometimes used asa reagent for transfecting nucleic acids into mammalian cells and hasrecently been used for cellular uptake of QDs. Duan et al. synthesizeddendritic PEG-grafted PEI ligand molecules and used them tofunctionalize CdSe—CdS—ZnS QDs (see reference 38). They demonstratedthat this specific combination of surface chemistries could mediate bothendocytosis and subsequent cytosolic delivery of these QDs to HeLacells. They found that the efficiency of QD endosomal escape wasenhanced by increasing the PEI content in the PEG-PEI ligand. However,the increased PEI content was also coupled with significant cytotoxicityas cellular viability dropped to only 40% with this more efficientendosomal escape capping ligand. The combination of PEG and PEI wastested for cellular uptake by complexing 520 nm QDs capped with 1:1DHLA:DHLA-PEG ligands with increasing ratios of PEI (˜0.5-10 μg PEI per1 pmol QD) and then exposing them to HEK 293T/17 cells for 1-3 h. Asdemonstrated by the representative micrograph in FIG. 4E, completecolocalization of the QDs with the transferrin marker within endosomeswas noted at each concentration tested even after several days.Similarly, a considerable degree of cytotoxicity was noted that trackedthe increasing concentrations of PEI used to form the QD-PEI complexes.

Jablonski et al. reported the ability to rapidly deliver polyargininepeptide-bearing QDs directly to the cytosol using an excess of thehydrophobic counterion, pyrenebutyrate (see reference 39). Usingstreptavidin conjugated QDs assembled with biotinylated polyargininepeptides in the presence of >1,000 fold molar excess pyrenebutyrate (4μM) per QD (4 nM), delivery of the QDs (˜5 min) to BS-C-1 monkey kidneycells appeared to produce QDs dispersed within the cytosol. Thoseauthors surmised that the anionic pyrenebutyrate bound to the cationicpolyarginine to form a hydrophobic polymeric complex that could passdirectly through the plasma membrane. However, when HEK 293T/17 cellswere incubated with 550 nm DHLA-PEG QD-CPP assemblies (100 nM in QDs) inthe presence of a far higher concentration of pyrenebutyrate (100 μM),the QDs initially remained entirely associated with the plasma membraneeven after a ˜6× longer incubation of 30 min. After 3 hours, the QDstook on a punctate appearance, indicative of endosomal uptake and nocytosolic dispersal was observed (see FIG. 4F). Thus, the resultsreported Jablonski et al. (reference 39) cells were not reproduced bythe present inventors. It remains unclear what role pyrenebutyrate playsin the uptake process, but these results suggest it is likely thatdifferences in the supplied material (i.e., presence or absence of acovalently attached 60 kDa streptavidin protein along with differentsurface ligands) may actually play profound part in determining thenature of the interaction of nanomaterials with the plasma membrane.

Another molecule was tested in this class of materials, namely thecommercially available PULSin™, a proprietary amphiphilic polymeroriginally designed as a cytosolic delivery agent for proteins. It wasfound that PULSin™ could mediate efficient uptake of QDs and a modestsubsequent endosomal release to the cytosol over a much longer timeperiod of 3-4 days. As shown in FIGS. 5A and 5B, PULSin™ complexationresulted in initial endosomal uptake of 520 nm DHLA:DHLA-PEG QDs to bothCOS-1 and HEK 293T/17 cells after 1d incubation. Following uptake, thecells were allowed to grow continuously for 5 days and samples wereimaged throughout. Approximately three to four days after the initialdelivery, the QD signal began to separate from that of the endosomalmarker, indicating endosomal escape in both cell lines. By day 5, theQDs had assumed a slightly more dispersed intracellular appearance thatwas completely distinct from that of the endosomes which had a moreperinuclear localization. Longer incubation times did not improve onthese results. The results were rather modest in terms of overallcellular labeling efficiency, but it was clear that combining PULSin™with mixed surface QDs could facilitate some endosomal escape. However,in addition to requiring several days to mediate endosomal escape of theQDs, PULSin™ also elicited a considerable degree of cytotoxicity in bothcell lines (FIGS. 5C and D). It was found that the toxicity wasattributable to the PULSin™ polymer alone. When cells were incubatedwith 100 nM mixed surface QDs alone, the viability of both COS-1 and HEK293T/17 cells was approximately 80% while in the presence of eitherPULSin™ alone or QD-PULSin™ complexes, cell viability was reduced toless than 60%.

Peptide-mediated delivery. The use of peptides still remains the mostpopular means of facilitated QD delivery (see reference 10). In thiscase, the QDs are decorated with a peptide that induces endocytosis bymediating interaction with specific cell surface receptors or moregenerally through electrostatic interactions with the cell surface (seereference 40). For electrostatic interactions, peptides derived from theHIV-1 Tat protein have been used.

Sucrose and chloroquine are two well known endoosmolytic agents thathave been shown to facilitate the release of endocytosed nucleic acidsto the cytosol (see reference 43). Sucrose accumulates within endocyticvesicles and promotes vesicle swelling and destabilization (seereference 44). Chloroquine is an endolysosomal-tropic amine whosebuffering capacity prevents endosomal acidification and slows down therate of endocytosis, allowing more time for endosomal escape (seereference 45). In this instance, 510 nm DHLA-PEG QDs were self-assembledwith CPP and incubated with HEK 293T/17 cells for 2-3 h in the presenceof increasing concentrations of either agent up to a maximum of 500 mMsucrose or 500 μM chloroquine. Both compounds efficiently disrupted theendosomes at the highest concentrations as evidenced by the diffuseappearance of labeled transferrin in the cytosol. The QDs, however,remained punctate in appearance and were not well-dispersed despite thepresence of the far more soluble PEGylated QD ligands (see reference18). Also noted were significant cellular toxicity and morbidity,especially as the concentrations of each agent were increased.

Nuclear localization signal (NLS) peptides bearing a sequence derivedfrom the simian virus 40 T-antigen have been reported to mediate QDuptake and subsequent nuclear delivery (see reference 46). Rozenzhak andco-workers utilized commercial Streptavidin functionalized QDs assembledwith biotinylated NLS peptides and incubated with a secondarynon-covalent peptide carrier (Pep-1) to create complexes for delivery toHeLa cells. They found a high degree of subsequent QD colocalizationwith the nuclear stain DAPI and little residual cytosolic or endosomalstaining. Thus, in a conjugation approach similar to that used for theCPP, as described herein a histidine-tagged NLS peptide was assembledonto the surface of QDs for delivery experiments. When incubated withHEK 293T/17 and COS-1 cells, however, the QDs were found to becompletely internalized within endosomes, with no release to the cytosolor nuclear translocation observed even after culturing the cells for twodays after QD uptake.

Furthermore, the use of a multifunctional peptide including a portionoriginally intended for the delivery of protein palmitoyl transferase 1(PPT1) inhibitors across the blood-brain barrier was tested (seereferences 47 and 48). This peptide, WG(Pal)VKIKKP₉GGH₆ (referred to asPalm-1 and having SEQ. ID. No. 3), encompasses several different modulesor domains each of which provides one or more overlappingfunctionalities. The peptide includes a His₆ tag to mediateself-assembly to the QD surface. The di-glycine residues were placedinto the sequence to act as a flexible spacer between the His₆ domainand the rest of the peptide once it coordinates to the QD surface.Adjacent to the polyhistidine domain is a repeat of nine consecutiveproline residues. Studies have shown that poly(L-proline) sequencesadopt a left-handed helical structure of 3.1 residues per turn inaqueous solvent, referred to as the polyproline II or PPII motif (seereference 49). Nine prolines are predicted to assume a PPII structurewith a length of ˜28-30 Å, thus this sequence was utilized as anextended spacer to allow the rest of the peptide sequence to extend outbeyond the PEGylated shell surrounding the QD (assumed length of ˜30-35Å in an energy minimized state). PPII motifs also possess amphiphilicproperties. Peptides containing proline repeats of various lengths havebeen used in relation to the cellular uptake of attached fluorophores(see references 50-52), DNA-lipid complexes (see reference 53) and goldnanoparticles (see reference 54). The VKIKK motif (SEQ ID No: 7) isderived from a sequence found at the C-terminus of the K-Ras protein(V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) (see reference55). This Ras family homologue is a membrane-tethered GTPase thatfunctions in many overlapping signal transduction pathways. The KKIKportion is thought to mediate electrostatic interactions with lipid headgroups on the cytosolic leaflet of membranes displaying negativelycharged phosphatidylserines. Similar to the positive charges residentwithin the Tat and NLS sequences, the three positively charged lysinesmay also facilitate the peptide's initial electrostatic interaction withnegative charges on the cellular membrane. In the K-Ras protein, theVKIKK (SEQ ID No: 7) sequence is preceded by a cysteine residue that isnormally post-translationally modified with a palmitoyl group which canalso be depalmitoylated by PPT1 in the lyososome. Due to its highlyhydrophobic nature, the palmitoyl group inserts into the membraneallowing the full K-Ras (or similar post-translationally modifiedproteins), to remain membrane-bound (see references 56-58). Rather thanattaching the palmitoyl group in the peptide to a cysteine residue whichwould leave it vulnerable to PPT1 cleavage in the endolysomal system, itwas decided to covalently attach it to a synthetic diaminopropionic acid(Dap) residue, thus rendering it nonhydrolyzable. It was conceived thatby using a peptide incorporating multiple overlapping functionalsequences shown to facilitate both endocytosis and intracellularmembrane interactions, cytosolic delivery of the QDs could be achieved.

COS-1 and HEK 293T/17 cells were incubated for 1-2 h with 550 nm DHLAPEG QD-Palm-1 complexes in the presence of AlexaFluor 647-transferrin.Imaging the cells at 1 hr post delivery revealed that the QD complexeswere internalized with a punctate appearance that was completelycolocalized with the endosomal marker confirming endocytic uptake. Asshown in FIGS. 6A and B, approximately 48 h post-delivery the QD signalseparated from that of the endosomal marker in both cell lines and theQDs became well-dispersed, occupying the entire cell volume. Greaterthan 90% of cells observed were positive for initial QD uptake and ofthose, 77% showed a high degree of endosomal escape. Although a smalldegree of endosomal escape was observed at 24 h, QD release fromendosomes was maximal at 48 h post-delivery and no appreciable increasein escape efficiency was noted at 72 h or longer. Some punctate areas ofbrighter QD fluorescence remained which were still colocalized with thetransferrin, leading us to conclude that not all endosomal QDs werereleased with this peptide.

Two further Palm-1 peptide variants were tested; one bearing only asingle proline residue and another having no proline residues, termedPalm-2 and Palm-3: in both cases, these resulted in a significantreduction in the degree of endocytic uptake and the QDs remainedcompletely sequestered within endosomes over similar 72 h observationperiods (see below for comparison of these Palm peptide sequences). Thissuggests that while the PPII motif may play a contributing role inmediating more efficient QD uptake by endocytosis, it is absolutelynecessary for promoting QD release to the cytosol in both cell lines.Studies in HeLa cells have pointed to a role for polyproline domains inmediating the uptake and cytosolic delivery of peptides containing acombination of proline repeats and various fatty acyl moieties (seereference 51), both features that are present within the Palm-1 peptide.However, the specific mechanism by which the polyproline domain withinthe Palm-1 peptide mediates uptake and eventual endosomal escape remainsunclear. It may provide these critical functions by serving as a spacerthat extends the VKIKK sequence (SEQ ID No: 7) and the palmitoyl groupbeyond the PEG surface, by contributing amphiphilic properties, somecombination of both, or by another as yet unidentified role.Significantly, it was found that the QD-Palm-1 complexes elicitedminimal cytotoxicity following uptake and endosomal escape, as seen inFIGS. 6C and 6D. In both COS-1 and HEK 293T/17 cells, at the QD-Palm-1complex concentration required for efficient cytosolic delivery (100nM), cell viability was greater than 85% even after 3 days. This valueis quite close to the minimal cytotoxicity that was noted for deliveringQD-CPP complexes to cells with short 1 hr incubations in previous studywhere the QDs remained sequestered within the endosomes (see reference13). Thus, although the QDs are escaping from the endosomes with highefficiency, the finding of low concomitant cytotoxicity suggests thatthe integrity of the endolysosomal system is not being compromised inthe process and adversely affecting metabolism or viability. Anothernoteworthy finding from these data is the reduced cytotoxicity of thepeptide-free QDs in HEK 293T/17 cells when their surface is cappedexclusively with DHLA-PEG ligands (greater than 90% cell viability, FIG.6D) compared to when a 1:1 surface of DHLA:DHLA-PEG ligands is used(˜75% cell viability, FIG. 5D). COS-1 cells, however, did not exhibitthis differential cytotoxicity response to the two QD surfaces (FIG. 6Cvs. 5C). This result not only demonstrates the important role played bythe capping ligand in mediating QD biocompatibility but also points toinherent differences between the two cell lines.

Comparison of several Palm peptide sequences. Described herein aremodular, multifunctional peptides that (1) assemble with nanoparticles(NP) to generate NP-peptide bioconjugates and (2) mediate the cellularuptake and intracellular distribution of the NP-peptide bioconjugates.The peptides are modular and multifunctional in nature, consisting ofdistinct domains wherein each domain performs a specific function withinthe NP-peptide assembly. The domains comprise: (1) an NP associationdomain which mediates the interface of the peptide with the NP surface;(2) a spacer domain to reduce steric hindrance between the peptide andthe NP surface; (3) a cellular association/uptake domain (sometimesreferred to as the uptake domain) that mediates the interaction of theNP-peptide assembly with the cell membrane and its components (e.g.,cell surface receptors) and the internalization of the assembly by thecellular machinery and (4) a “vesicle escape” domain that mediates theescape of the NP-peptide assemblies from within intracellular vesiclesof the cellular uptake pathway, resulting in the intracellulardistribution of the NPs.

As an example, a multifunctional, modular peptide was used to mediatethe intracellular delivery and the ultimate endosomal release ofluminescent semiconductor nanocrystals or quantum dots (QDs). Thepresent disclosure is generally applicable to other classes of NPs andnanomaterials (e.g., gold NPs, carbon nanotubes) and is not limited tothe decoration of the NP surface with only a single multifunctionalpeptide species. The generation of NPs bearing ensembles of differentpeptides wherein each peptide imparts a different biological function isenvisioned.

Potential uses of the peptides described herein include applicationareas where multifunctional NP-peptide assemblies might be employed,including both biological and nonbiological applications. Theseapplications include (but are not limited to) NPs assembled withmultifunctional peptides for the purposes of: (1) general cellularlabeling, (2) labeling of specific subcellular compartments, (3)cellular tagging for the purposes of cell sorting and/or cataloging, (4)drug delivery and drug monitoring, and (5) in vivo imaging. Thedecoration of nanoparticles (NP) with polymeric molecules (e.g.,proteins, peptides, nucleic acids) has been employed extensively tocreate hybrid functional materials in which the hybrid assembly adopts afunctionality that is greater than either of the two individualcomponent materials alone. This “value-added” approach has resulted inthe production of nanoparticle assemblies for non-biologicalapplications including chemical sensing and molecular electronics. Morerecently this approach has been used for biological applicationsincluding the in vitro sensing of enzymatic activity and the labeling ofprokaryotic and eukaryotic cells. In these approaches, the polymer usedto decorate the NP surface has typically been monofunctional in nature(e.g., an antibody that binds to its cognate ligand or a peptide thatserves as a substrate for a targeted enzyme). Descriptions of the use ofrationally-designed, multifunctional polymers, particularly for thepurpose of cellular uptake and the subsequent release from intracellularvesicles, have not been described. To achieve the delivery of NPs intocells, a class of peptides known as cell penetrating peptides has beenemployed previously with success. These peptides, often based on theTat-1 peptide of HIV, use the cells endocytic pathway to achieve thecellular uptake of the NPs. However, the NPs typically remainsequestered within the intracellular vesicles of the endocytic pathwayand they must be physically or chemically induced to be released intothe cell cytoplasm.

Described herein is a new approach for the functionalization ofconceivably any NP with a rationally-designed, modular, multifunctionalpeptide to generate NP-peptide assemblies capable of “escaping” fromwithin endocytic vesicles. The peptide carries out two main functions:(1) it mediates the cellular uptake of the NP to which it is attachedand (2) it mediates the release of the NP-peptide assemblies from withinintracellular endocytic vesicles. The peptide is comprised of separatefunctional domains that, while serving distinct roles. work together asan ensemble to impart multiple functionalities to the NP. In the exampleprovided, the NP is a semiconductor quantum dot (QD) and the peptide isa multi-domain peptide that mediates the cellular binding, uptake, andsubsequent intracellular dispersal of the QD.

FIG. 7A shows a general schematic representation of an exemplaryNP-peptide assembly, wherein the peptide comprises multiple functionaldomains: a NP association domain (1); a spacer domain (2): and multipledomains that impart biological activity (3 and 4). The multi-domainpeptide interfaces with the NP surface via the peptide's NP-associationdomain. This association can be through either a covalent bond or anoncovalent interaction (e.g., electrostatic or metal ion coordination).Subsequent functional domains are located adjacent from theNP-association domain. Such peptides can be synthesized or they can beexpressed and purified through molecular biology techniques known to aperson of ordinary skill in the art.

FIG. 7B shows an exemplary peptide attached to a QD and effective toprovide for intracellular uptake and endosomal escape. The modularpeptide consists of a 6-histidine (H6) domain for assembly to the QDsurface, a proline-rich spacer domain (P9GG), a positively chargedlysine-rich domain (VKIKK) for cellular association and uptake, and asynthetic palmitic acid group ((Pal) Dap, representingpalmitoyldiamoniproprionyl) for cell membrane association and insertion.The AcG(Pal)DapVKIKK portion of the sequence is derived from theC-terminus of the K-ras protein where the (Pal)Dap is a nonhydrolyzablemimic of a palmitoylated cysteine residue. This sequence has been shownto inhibit a palmitoyl thioesterase with low micromolar affinity inacidic environments. See G. Dawson et al., Biochemical and BiophysicalResearch Communications, 395 (2010) 66-69. It is believed that thisprotects the palmityl group from endosomal degradation. The domains actin concert to impart desirable biological capabilities to the QD.(Pal)Dap is sometimes referred to as Dap(Pal).

Numerous different multidomain, multifunctional peptides have beenassembled to the surface of QDs. They can be monitored the intracellularfate of the different QD-peptide assemblies both in terms of the abilityof the assemblies to be internalized by cells and to be liberated fromwith in intracellular vesicles (endosomes). To this end, threestructurally similar, modular peptides were synthesized: Palm-1b,Palm-2b, and Palm-3b.

As compared to Palm-1 discussed above, the Palm-1b sequence lacks theterminal tryptophan which in Palm-1 serves to provide an aromatic ringstructure for monitoring by absorption spectroscopy. It also usesN-acetylglycine in place of glycine. It is not believed that thesedifferences result in any change in function with regard to ability tomediate delivery of nanoparticles.

The tested peptide sequences are shown below in Table 2. The threepeptides share in common the hexa-histidine domain for assembly to a QDsurface, the positively-charged lysine-rich domain (VKIKK) (SEQ ID No:7) for cellular association and uptake, and a synthetic palmitic acidgroup ((Pal)Dap, representing palmitoyldiamoniproprionyl) for cellmembrane association and insertion. The three peptides differ, however.in the sequence of their spacer domains. The spacer domain of Palm-1 hastwo glycine residues followed by a nine proline repeat and is predictedto adopt a rigid, extended helical conformation based on the fixed sidechain conformation of the successive pyrrolidine rings of the prolineresidues. The Palm-2b and Palm-3b peptides are glycine- and alanine-rich(with Palm-3b having one proline residue substituted for an alanine) andare expected to adopt shorter alpha-helical conformations relative tothe proline helix.

TABLE 2 Multifunctional modular peptides for cellular uptake PeptideSequence Sequence Number Palm-1b AcG(Pal)DapVKIKK P₉GG H₆ SEQ ID NO: 4Palm-2b AcG(Pal)DapVKIKKGLAAAAGGH₆ SEQ ID NO: 5 Palm-3bAcG(Pal)DapVKIKKGLAPAAGGH₆ SEQ ID NO: 6 Pal, palmitoyl; Dap.diaminopropionic acid; Ac, acetyl group blocking the peptide'sN-terminus.

Palm-1b, Palm-2b, and Palm-3b have approximate molecular weights of 3992Da, 3570 Da, and 3594 Da, respectively.

Molecular modeling was employed to examine the effects of the differentspacer domain sequences on the conformation of the peptides within theQD-peptide assemblies, as seen in FIG. 8, which shows the resultingoverlaid QD-peptide models obtained when each peptide was energyminimized for its association with the QD surface via its 6-histidinedomain. Shown is are comparative models of each of three Palm peptidesassembled onto the surface of a QD bearing a polyethylene glycol-600(PEG-600) surface coating. The Palm-1b peptide is shown in medium-graywhile the Palm-2b and Palm-3b peptides are shown in white and dark gray,respectively. Each peptide is shown assembled with the QD surface shellvia its 6-histidine domain. The spacer domains of the Palm-2b and Pal-3bpeptides adopt a short alpha-helical conformation that allows only aportion of the lysine-rich domain and the palmitic acid group toprotrude from the PEG-600 layer. The proline repeat-containing spacer ofPalm-1, however, adopts a rigid, more extended helical conformation thatpositions both the lysine-rich cell-binding domain and the palmitic acidgroup well beyond the PEG layer, making them readily accessible to thecell surface. The spacer extends about ˜30 Å through the PEG-600 layerand presents the lysine-rich cell-binding domain and the palmitic acidgroup in an accessible orientation beyond the PEG layer. The extendedspacer domain of Palm-1b positions the palmitic acid group approximately22 Å further away from the PEG layer relative to the Palm-2b and Palm-3bpeptides.

The functional implications of the different conformations of the Palmpeptides with in the QD-peptide assemblies were assessed by celldelivery experiments, as seen in FIG. 9. In FIGS. 9A, B, and C, theassembles have Palm-1b, Palm-2b, and Palm-3b, respectively.

The peptide assemblies were incubated with COS-1 cells for 1 hr,removed, and the cells were subsequently cultured for 24 hr prior tofixation. An AlexaFluor 647-transferrin (Tf) conjugate was includedduring the incubation to label the vesicles of the endocytic pathway.Cell nuclei were stained with DAPI. Upon internalization, the Palm-1bQD-peptide assemblies were largely well dispersed throughout thecytoplasm and were not colocalized with the endocytic marker. ThePalm-2b and Palm-3b QD assemblies were nearly entirely sequesteredwithin endosomes.

The intracellular fate of the three different QD-peptide assemblies wasmonitored after incubation with COS-1 cells by noting the distributionof the assemblies within the cell and by labeling the endocytic pathwaywith a marker specific for endosomes (AlexaFluor 647-conjugatedtransferrin). As shown in FIG. 9, each of the three peptides mediatedintracellular uptake of the QD-peptide assemblies. A notable differencewas observed in the intracellular distribution of the assemblies. ThePalm-2b and Palm-3b assemblies remained completely sequestered withinendocytic vesicles as evidenced by their colocalization with theendosomal marker. The Palm-1b QD-peptide assemblies, however, werewell-dispersed throughout the cytoplasm while only a small percentagewas colocalized within endosomes. Thus, the proline-rich spacer domainof Palm-1b in conjunction with the other modular peptide propertiescombine to mediate both cellular uptake and endosomal escape of theQD-peptide assemblies.

Domains of a peptide for delivery of nanoparticles. A peptide fordelivery of a nanoparticle to the cytosol comprises: (a) a nanoparticleassociation domain; (b) a spacer domain; (c) an uptake domain; and (d) avesicle escape domain comprising a non-hydrolyzable lipid moiety.

One of ordinary skill in the art may configure the nanoparticleassociation domain to make the peptide amenable to either covalent andnoncovalent attachment strategies. For example, the nanoparticleassociation domain can be a poly-histidine sequence which noncovalentlybinds metal in a payload, such as a metallic quantum dot. Apolyhistidine domain could also be used to bind anickel-nitrilotriacetic acid (Ni-NTA) linker, where the linker can beattached to a payload such as a drug. The nanoparticle associationdomain could also be adapted for coupling to a payload using1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) forcrosslinking a carboxyl groups to a primary amine. Other possibleconjugation protocols for the nanoparticle association domain are knownto one of ordinary skill in the art, and include maleimide dithiolexchange, diazonium salt chemistry, and the like. In another embodiment,the nanoparticle association domain comprises a plurality of eitherpositively- or negatively-charged amino acid residues, so that thepeptide is electrostatically attracted to a payload having an opposingcharge.

The spacer domain serves to reduce to reduce steric hindrance betweenthe peptide and the surface of the nanoparticle, and may have otherfunctions. Optionally, the spacer has a length 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 proline residues. In an embodiment, the spacer comprises apoly-proline repeat. In another embodiment, the spacer comprises apoly-arginine repeat. In yet another embodiment, the spacer comprises anumber of synthetic peptides, such as Aib (alpha-amino isobutyric acid).

The uptake domain is preferably a positively charged lysine-rich domainsuch as VKIKK (SEQ ID No: 7).

The vesicle escape domain preferably comprises a non-hydrolyzable lipidmoiety. Such a lipid moiety includes at least eight carbons. Optionally,there may be more than one such moiety, for examplediamoniproprionyl-glycine-diamoniproprionyl. In one embodiment, thelipid moiety is palmitate (palmitic acid). One of ordinary skill in theart would recognize that another lipid moeity might be used to obtainthe desired cell membrane association, insertion, and/or vesicle escape.Such moieties include octanoic, 2-ethylhexanoic, nonanoic, decanoic,lauric, myristic, margaric, stearic, arachidic, behenic, lignoceric acidor unsaturated monocarboxylic acids such as oleic, linoleic, linolenic,ricinoleic acid or aromatic monocarboxylic acids such as benzoic acid,aliphatic dicarboxylic or polycarboxylic acids such as succinic,glutaric, adipic, pimelic, suberic, azelaic, sebacic,nonanedicarboxylic, decanedicarboxylic, dimer fatty acids, which areobtainable by dimerizing unsaturated monocarboxylic acids; aromaticdicarboxylic or polycarboxylic acids such as terephthalic, isophthalic,o-phthalic, tetrahydrophthalic, hexahyrophthalic or trimellitic acid,for example. Other such moieties include cholesterol. Also included inthe definition of lipid moeity as used herein areperfluorocarbon-containing compounds having at least eight carbons. Mostpreferable, the lipid moiety is non-hydrolyzable.

Modular design of functional domains allows for flexibility in iterativepeptide development and testing. Further experiments were conducted toinvestigate the roles of these domains. The tested peptides (includingcontrols) and the results of the experiments are shown in Table 3.

TABLE 3 Additional Peptides Tested SEQ ID Peptide Sequence DescriptionNo: JB577 WGDap_(a)VKIKK(P)9GG(H)6 Endosomal escape 2 (Palm-1) peptidePalm 2 WGDap_(a)VKIKKGL(A)4GG(H)6 No (Pro)9, short linker 9 Palm 3WGDap_(a)VKIKKGLPAAGG(H)6 No (Pro)9, shorter linker 10 JB582WGDap_(a)VKIKK(P)4GG(H)6  Shorter (Pro)4 linker 11 JB747WGDap_(a)VKIKK(Aib)9GG(H)6 (Pro)9 replaced with 12 (Aib)9 JB865WGDap_(a)VKIKK(P)12GG(H)6 Longer (Pro)12 linker 13 JB864WGDap_(a)VKIKK(P)15GG(H)6 Longer (Pro)15 linker 14 JB578WGDap_(a)(P)9GG(H)6 No Lysine residues 15 (uncharged) JB583WGDap_(a)VRRRIRR(P)9GG(H)6 Arg substituted for Lys 16 JB585WGDapVRRRIRR(P)9GG(H)6 Arg substituted for Lys, 17 no palmitate JB833WGDap_(a)AGAGG(A)4Aib(A)4GG(H)6 Uncharged 18 Palm-only JB869WGDap_(a)VRL(P)3VRL(P)3VRL(P)3GG(H)6 Amphipathic repeat 19places VKIKK(P)9 JB868 WGDapVRL(P)3VRL(P)3VRL(P)3GG(H)6Amphipathic VRL(P)3 20 3-repeat; no palmitate JB579 WGDapVKIKK(P)9GG(H)6No palmitate group 21 JB580 WGDap_(a)GDapaVKIKK(P)9GG(H)6 Two palmitates22 JB866 WGDap_(aa)VKIKK(P)9GG(H)6 Two palmitates on Dap 23 JB589WGCcVKIKK(P)9GG(H)6 Hydrolyzable palmitate 24 JB858WGDab_(a)VKIKK(P)9GG(H)6 Dab replaces Dap 25 JB641WGDap_(di)VKIKK(P)9GG(H)6 8-carbon alkane group 26(ontanoyl) substituted  for palmitate JB621W_(d)GDap_(di)VKIKK(P)9GG(H)6 Two octanoyl groups 27 JB729WGDap_(e)VKIKK(P)9GG(H)6 Highly-fluorinated group 28substituted for palmitate SI-09160 WGDap_(f)VKIKK(P)9GG(H)6Cholesterol substituted 29 for palmitate JB876 WGC_(f*)VKIKK(P)9GG(H)6Modified cholesterol 30 on cys JB872 WGC_(g)VKIKK(P)9GG(H)6Farnesyl replaces 31 Palmitate JB859 (H)6GG(P)9WGDap_(a)VKIKKReversed sequence 1 32 JB860 (H)6GG(P)9VKIKKDap_(a)GWReversed sequence 2 33 JB862 VKIKKWGDap_(a)(P)9GG(H)6Reversed sequence 3 34 JB588 WGDap_(a)RRRIRR(P)9GGK No (His)6, for EDC35 coupling JB728 WGDap_(a)VKIKK(P)9GGK_(b) Dye-labeled Enx1 36 JB722_(bi)KWGSAibAAALGG(R)10 Dye-labeled cell 37 penetrating peptide JB719KETWWETWWTEWSQPKKKRKVSGAibAAAGG(H)6 Chariot peptide 38 JB532 C(P)9GG(H)6Texas-Red (Pro)9 39 peptide (FRET acceptor) JB434 (R)9GGLAAibSGWK(H)6cell penetrating peptide 40 JB867 K_(h)GDap_(a)VKIKK(P)9GG(H)6Enx1 w/N-terminal TAMRA 41

In Table 3, “Dap” represents 2,3-Diaminopropionic acid, which is alsocalled “Dpr.” “Dab” represents diaminobutyric acid. “Aib” refers toalpha-amino isobutyric acid. Subscripts within the sequences refer tothe moieties depicted in FIG. 10.

Results of tests of update and endosomal escape of these peptides inCOS1 cells are provided in Table 4.

TABLE 4 Results of Tests on Additional Peptides # of % QD % endosomalescape % Escape in cells Overall Peptide (description) cells (n) uptake(n) of total cells (n) with uptake uptake/escape¹ JB577 (Palm-1) 76 92%(70) 69% (52) 77% High/high JB578 (Palm-1 159  8% (13) 4% (6) 46%Low/moderate uncharged) JB579 (Palm-minus) 82 17% (14) 5% (4) 29%Low/low JB580 (Palm-1 X2) 90 13% (12) 12% (11) 92% Low/high JB582(Palm-1/4 Pro) 67 0% (0) 0% (0) n/a None/none JB583 (Palm-1/R for K) 15519% (29)  8% (12) 41% Low/moderate JB585 (Palm-minus/R for 199 2% (4)0.5% (1)  25% Low/low K) JB589 (Palm- 144 6% (8) 3% (4) 50% Low/moderate1/hydrolyzable) JB833 (uncharged with — Not —  — — Palm) available JB858(Dab for Dap) — Membrane —  — — localized JB859 (Reverse order, 180  57%(103) 10% (19) 17% Moderate/low palm before VKIKK) JB862 (VKIKK moved)163 21% (35) 3% (3)  9% Low/low JB866 (2 Palm groups on 197 45% (89)  5%(10) 11% Moderate/low Dap) JB621 (Octanoyl X2) 241 29% (69) 26% (62) 90%Low/high JB641 (Octanoyl) 165 50% (83) 44% (73) 88% Moderate/high JB747(Palm-1/Aib for P) 78 100% (78)  58% (45) 58% High/moderate JB729 (Palm-80 64% (51) 54% (43) 84% High/high minus/fluorine) SI09160 (Palm- 10244% (45) 42% (43) 96% Moderate/high minus/cholesterol) JB860 (Reverseorder) 164 30% (50) 18% (30) 60% Moderate/moderate JB865 (P12 for P9) 8280% (66) 85% (56) 68% High/moderate JB864 (P15 for P9) 99 82% (81) 70%(69) 85% High/high JB872 (Palm- 101  99% (100) 78% (79) 79% High/highminus/farnesyl) JB869 (3 Amphipathic 315  80% (253)  44% (139) 55%High/moderate VRLPs for P) JB868 (3 Amphipathic 171  59% (101) 11% (19)18% Moderate/low VRLPs for P, no palm) JB876 (Cholesterol for — Notpalm, Cys linkage) available JB867 (Palm/TAM -    >90% integrity)QD/TAMRA colocalized JB588 (EDC conjugated) 64 94% (60) 80% (51) 85%High/high JB728 (Palm-1 84 81% (68) 76% (63) 93% High/high Rhodamine)JB719 (Chariot peptide) 180 53% (96) 23% (41) 43% Moderate/moderateJB722 (CPP Rhodamine) 94 13% (12) 8% (1)  1% Low/low

The Palm-1 peptide was also tested in HeLa cells, and found to be veryeffective in promoting endosomal escape. From a collection of 163 HeLacells administered Palm-1 conjugated to a quantum dot (QD), 90% (147)demonstrated QD uptake, 77% (126) of the total demonstrated endosomalescape, so that 86% of the cells with uptake demonstrated endosomalescape.

As a further example, a peptide having the described domains wassuccessful in delivering the 240-kDa B-phycoerythrin protein to thecellular cytosol.

The described peptides could be expressed using recombinant technology,optionally together with one or more additional peptide or proteindomains to provide further functionality. Such functionality can includetherapeutic functions. For example, one of ordinary skill could designan expression vector for transient and/or stable expression in a cell,tissue, or organism of a protein incorporating one or more of thedescribed peptides.

The strategy described herein is amenable to the assembly of a widerange of nanoparticles with a modular, multifunctional peptide. The NPsurface can be functionalized with different types of modular,multifunctional peptides (“mixed” surfaces), giving ratiometric controlover the nature of the decorated NP surface. Specifically for cellpenetrating peptide-mediated NP uptake, a peptide sequence that caneither individually or cumulatively: inherently interact with/attachitself to the NP surface; is amenable to having its lateral extension orrigidity controllably adjusted; contains a cell-penetrating domain;contains an endocytic vesicle escape sequence; potential to express orcontain other modular functional domains as desired; and the ratio orvalence (and thus the avidity) on the NP can be controlled.

In conclusion, described herein are peptides that include multipleoverlapping functional domains, the combination of which contributes toboth endocytosis and subsequent release to the cytosol of either thepeptide itself or the peptide attached to a nanoscale payload. Suchpayloads include proteins, carbohydrates, and other biologicalmaterials, drugs, contrast agents (or other imaging agents), nucleicacids, and/or gene therapy agents.

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1. A peptide for delivery of a nanoparticle to the cytosol, the peptidecomprising: (a) a nanoparticle association domain; (b) a spacer domain;(c) an uptake domain; and (d) a vesicle escape domain comprising anon-hydrolyzable lipid moiety, wherein the spacer domain is between thenanoparticle association domain and the uptake and vesicle escapedomains, and wherein the peptide, when attached to an extracellularnanoparticle, is effective to induce uptake of the nanoparticle by acell and delivery of the nanoparticle to the cytosol of the cell.
 2. Thepeptide of claim 1, wherein the spacer domain consists of from six tofifteen proline residues.
 3. The peptide of claim 1, wherein the vesicleescape domain comprises a non-hydrolyzable lipid moiety or perfluorogroup.
 4. The peptide of claim 3, wherein said moiety ispalmitoyldiamoniproprionyl.
 5. The peptide of claim 1, wherein thenanoparticle association domain comprises polyhistidine.
 6. A quantumdot attached to a peptide of claim
 1. 7. A quantum dot attached to apeptide of claim 1, wherein the peptide nanoparticle association domaincomprises poly-histidine.
 8. The peptide of claim 1 attached to ananoparticle, wherein the nanoparticle is selected from the groupconsisting of a protein, a polymer, a dendrimer, a carbon nanotube, arod, a sphere, a metallic particle, and combinations thereof.
 9. Apeptide comprising: (a) a nanoparticle association domain comprisingpolyhistidine; (b) a spacer domain comprising 8, 9, or 10 prolineresidues; (c) an uptake domain comprising SEQ ID No: 7; and (d) avesicle escape domain comprising a non-hydrolyzable palmitic acid,wherein the spacer domain is between the nanoparticle association domainand the uptake and vesicle escape domains.
 10. The peptide of claim 9,wherein the vesicle escape domain comprising non-hydrolyzable palmiticacid is palmitoyldiamoniproprionyl.
 11. The peptide of claim 9, whereinthe spacer domain comprises 9 proline residues.
 12. The peptide of claim9, wherein the peptide comprises SEQ ID No: 3 or SEQ ID No:
 4. 13. Amethod of delivery of a nanoparticle to the cytosol of a cell, themethod comprising providing to a cell a nanoparticle attached to apeptide according to claim 1, and allowing the cell to take up thenanoparticle.
 14. The method of claim 13, wherein the nanoparticle is aquantum dot.
 15. A method of delivery of a nanoparticle to the cytosolof a cell, the method comprising providing to a cell a nanoparticleattached to a peptide according to claim 8, and allowing the cell totake up the nanoparticle.
 16. The method of claim 15, wherein thenanoparticle is a quantum dot.
 17. A method of delivery of ananoparticle to the cytosol of a cell, the method comprising providingto a cell a nanoparticle attached to a peptide according to claim 11,and allowing the cell to take up the nanoparticle.
 18. The method ofclaim 17, wherein the nanoparticle is a quantum dot.
 19. A nucleic acidencoding a peptide of claim 1.